The p38 MAP kinase is involved in the fruit fly’s biological clock

Although p38 MAPK is expressed in the hamster SCN [41] and regulates the chick pineal circadian clock [42], expression in the fly's clock has not been reported so far. The endogenous clock of Drosophila consists of approximately 150 clock neurons in the brain that are largely subdivided into 9 subgroups: small ventral lateral neurons (s-LN_vs), large ventral lateral neurons (l-LN_vs), 5^th small ventral lateral neuron (5^th s-LN_v), dorsal lateral neurons (LN_ds), 4 clusters of dorsal neurons (DN_1as, DN_1ps, DN₂s and DN₃s) and lateral posterior neurons (LPNs) [47]–[49]. To investigate whether the clock neurons utilize p38 MAPK signaling pathways, we did immunohistochemistry on adult brains using the enhancer trap line p38b-Gal4 in combination with a UAS-GFP transgene. GFP-expressing brains were immunolabelled with anti-GFP, anti-PER and anti-PDF at ZT21 (3 h before lights-on), when PER is mainly nuclear. Interestingly, p38b-driven GFP showed a broad expression within the brain as reported in Vrailas-Mortimer et al. [27] and colocalized with anti-PER and anti-PDF in at least four clock neurons, the large ventral lateral neurons (l-LN_v, Fig. S1). Although, we were not able to reliably co-stain more clock neurons, our p38b-Gal4-staining pattern suggests that p38 is likely expressed in further clock neurons. To verify this we performed p38 antibody staining on Canton S wildtype brains using three different antibodies – two raised against Drosophila p38 (not distinguishing between the isoforms and between active/phosphorylated and inactive/unphosphorylated p38) and one raised against the dually phosphorylated isoforms of human p38 recognizing also phosphorylated Drosophila p38 (Cell Signaling Technology). The two Drosophila p38 antibodies, p38b (kindly provided by T. Adachi-Yamada) and p38 (Santa Cruz Biotechnologies), gave rather broad staining with several cell bodies labeled in the region of the clock neurons resembling the staining pattern of the p38b-driven GFP (Fig. 1A–C; Fig. S1). Double-labeling with anti-VRI and anti-PDF showed that both antibodies reliably labeled the PDF-positive l-LN_vs as well as the PDF-positive s-LN_vs (as depicted for anti-p38b in Fig. 1A–C). In addition, there was staining in the entire cortex of the dorsal brain including the region of the dorsal neurons (Fig. S1). In comparison, immunostaining with phospho-p38 MAPK antibody (hereafter also referred as p-p38) also showed clear labeling in the protocerebrum (Fig. 1F), but p-p38 staining of clock neurons was restricted to much fewer cells. We found reliable staining only in the DN_1as (Fig. 1F, G) and in one experiment also in the l-LN_vs (not shown). This discrepancy might be due to the specificity of p-p38 antibody, which rather represents the current activation pattern than expression pattern of p38. Generally, tiny amounts of activated kinases are sufficient for effective signaling in transduction cascades. Thus, the amount of activated p38 might be well below the detection limit of the p-p38 antibody in the majority of clock neurons. In addition, p38 may be temporally phosphorylated as shown for the hamster SCN, where activated p38 was only high in the late day and early night [41]. Indeed, we noticed that p-p38 levels in the DN_1as as well as staining in the entire cortex of the brain depended also on the time of day and were only high towards the end of the night (Fig. 1D,E2 compared to Fig. 1F,G2).

To finally exclude any unspecific antibody labeling, antibody staining on two p38 null mutants, w¹¹¹⁸;+;p38a^Δ1 and yw;p38b^Δ45;+ (from now on referred to as p38a^Δ1 and p38b^Δ45, respectively), was performed at ZT21 and p-p38 staining intensity was measured in DN_1as. Both p38 mutants displayed a significant reduction in phosphorylated p38 to 50% of wildtype level (p<0.05; Fig. S2). This clearly verifies the authenticity of the p-p38 antibody labeling, but also suggests the existence of both p38 isoforms in these cells.

Taken together, even if we did not get a complete overlap, p38b-driven GFP expression as well as p38 antibody staining indicates that both p38a and p38b are expressed in several clock neurons, most probably in the PDF-positive l-LN_vs and s-LN_vs as well as in the DN_1as. This finding coincides with other studies: Microarray studies on LN_vs detected enriched p38a mRNA levels in the s-LN_vs as compared to other brain regions [50]. Furthermore, Mef2, a transcription factor well recognized as a downstream target of p38 MAPK signaling in Drosophila muscle [27] and mammalian myocytes, lymphocytes and neurons [29], [51]–[53], was shown to localize in all subgroups of Drosophila clock neurons [54] indicating p38 MAPK signaling in these cells.

So far, Drosophila studies mainly focused on p38 MAPK expression over a longer period of time, especially with regard to development [26], [37], [55]. Since the observed changes in the amount of phosphorylated p38 in the DN_1as at ZT9 and ZT21 (Fig. 1D–G) might also point to daily oscillating gene expression, we examined mRNA levels of p38a and p38b in the course of a day.

Quantitative real-time PCR (qPCR) from head extracts of Canton S wildtype flies revealed an allover higher expression level of p38b compared to p38a throughout the day (p<0.001; Fig. 2A). This is consistent with data published in a microarray-based atlas of gene expression in Drosophila (Flyatlas - http://www.flyatlas.org↗). Moreover, we did not discover any circadian oscillations of p38 isoforms on the transcriptional level, which is reminiscent on findings in fungi [44] and mammals [41], [56]. Very similar to our study, the latter papers demonstrated rhythmic phosphorylation of p38 throughout the day while total protein levels remained constant. This clearly indicates that activation and not expression of p38 is clock-controlled.

For studying oscillations in active p38 in more detail, immunohistochemistry on Canton S wildtype brains was carried out in LD 12∶12 at different times of day. Triple-labeling with anti-p-p38, anti-VRI and anti-PDF revealed daily oscillation in p38 phosphorylation in DN_1as, with low levels during the light phase (ZT1-9) and significantly higher levels in the dark (ZT13-21) (p<0.05; Fig. 2B). Furthermore, the average number of p-p38-positive DN_1as per hemisphere was significantly higher at night than during the day (p<0.05; Fig. S3). The diurnal oscillation in phosphorylated p38 in DN_1as strongly points to a clock-mediated activation of p38 within the circadian system. To test whether these diurnal variations in active p38 are indeed clock-controlled or just represent a direct response to darkness, p-p38 staining intensity in the DN_1as was measured under constant conditions at CT6 and CT18. Interestingly, similar to our observations in LD, the level of active p38 was significantly lower in the subjective day than subjective night confirming our hypothesis of a clock-controlled phosphorylation of p38 (p<0.001; Fig. 2C). Since previous studies in mice [57]–[59] and hamsters [41] also suggested a light dependent regulation of ERK and p38 activity in the SCN, we further exposed flies at CT6 and CT18 for 15 minutes to light and dissected brains before and after light pulse treatment. While levels of active p38 at CT 6 remained constant, light pulse at CT 18 led to a significant decrease in p-p38 signal (p<0.05; Fig. 2D). These results indicate an additional light-induced regulation (depression) of p38 activity.

Taken together, our findings are in strong favor of a clock-controlled phosphorylation of p38. Both p38a and p38b are constantly expressed throughout the day and display no circadian regulation on transcriptional level. Activation of p38 MAPK, however, seems to be clock-regulated, showing high levels of active p38 during the night and low levels during the day as we could show for the DN_1as. This would argue for a night-time specific function of p38 within the clock of these neurons. Nevertheless, we have to admit that the DN_1as are not the clock neurons that are most important for the control of behavioral rhythmicity. Future studies have to show, whether a cyclic activation of p38 does also occur in the s-LN_vs.

Locomotor activity recordings are a well-suited technique for investigating circadian behavioral rhythms in Drosophila melanogaster. When entrained to LD cycles wildtype flies display a typical bimodal activity pattern with an anticipatory morning and evening activity peak around lights-on and lights-off. In constant darkness this rhythmic locomotor behavior proceeds with its internal individual period reflecting the pace of the endogenous clock. To examine the role for p38 MAPK within the circadian system, we used transgenic RNA interference (RNAi) to reduce p38b RNA levels and thus p38b activity in different subsets of clock neurons, and screened for altered behavioral rhythms in LD as well as in constant dark conditions (DD). For RNAi-mediated p38b knockdown a w;UAS-p38b^RNAi;+ line was combined with different drivers as well as a UAS-dicer2;+;+ line (dicer2). We first used dicer2;tim(UAS)-Gal4;+, a driver line with a broad expression pattern that allows ubiquitous expression in all clock cells. Daily activity patterns of dicer2;UAS-p38b^RNAi/tim(UAS)-Gal4;+ flies were similar to those of control flies showing normal wildtype LD behavior with activity peaks around lights-on and lights-off (Fig. 3A). To test the effectiveness of p38b transgenic RNAi, we performed qPCR on head extracts and found no significant reduction in p38b mRNA level in our experimental line. This may be due to a small number of p38b-positive clock neurons compared with the total number of p38b-expressing neurons within the brain (Fig. S1 compared to Fig. 1). Thus, w;UAS-p38b^RNAi;+ was additionally combined with da-Gal4, a line that expresses Gal4 in most tissues throughout development [60]. Using the broader driver, we finally observed a significant decrease in p38b mRNA level in w;UAS-p38b^RNAi/+;da-Gal4/+ compared to respective controls, confirming the effectiveness of our p38b^RNAi construct (p<0.05; Fig. S4). Since we found no behavioral phenotype in LD, we next focused on locomotor behavior of dicer2;UAS-p38b^RNAi/tim(UAS)-Gal4;+ flies under constant conditions using χ²-periodogram analysis. Surprisingly, 93% of the flies were arrhythmic (Table 1) and only 7% showed rhythmic locomotor behavior with a prolonged free-running period of 25.3 h (p<0.001; Fig. 3A; Table 1). Considering the fact that besides clock neurons dicer2;tim(UAS)-Gal4;+ additionally drives expression in glia cells, we wanted to rule out a glia-specific effect on rhythmicity and period length. Therefore, we restricted p38b knockdown solely to the PDF-expressing clock neurons, the s-LN_vs and the l-LN_vs, using the more specific clock driver dicer2;Pdf-Gal4;+. Dicer2;UAS-p38b^RNAi/Pdf-Gal4;+ flies showed a later onset of evening activity and a higher activity after lights-off than control flies in LD (Fig. 3B) as well as a significantly prolonged free-running period of 24.8 h in DD (p<0.05; Fig. 3B; Table 1). Only about half of the flies were arrhythmic as opposed to 93% of dicer2;UAS-p38b^RNAi/tim(UAS)-Gal4;+ flies (Table 1). These findings suggest that p38 has indeed a functional role within the circadian system and that its specific knockdown in the clock neurons mainly delays evening activity and lengthens the free-running period.

To further confirm our hypothesis of p38 functioning in the clock three additional constructs were expressed to interfere with endogenous p38b: two UAS-p38b kinase-dead transgenes (UAS-p38b^KD3 and UAS-p38b^KD8) and a dominant-negative UAS-p38b transgene (UAS-p38b^DN-S). Interestingly, simultaneous expression of UAS-p38b^KD3 and UAS-p38b^KD8 in either PDF- or TIM-expressing neurons resulted in a delayed onset of evening activity and a prolonged free-running period under DD compared to respective controls, but did not cause any arrhythmicity (p<0.001; Fig. S5; Table 1). This phenotype became even more obvious when the dominant-negative p38b transgene was expressed in all clock cells (using w;tim(UAS)-Gal4;+) or only in the LN_vs (using yw;Pdf-Gal4;+): evening activity was delayed in LD and, in DD, free-running period was lengthened for about 2 h in UAS-p38b^DNS;tim(UAS)-Gal4/+;+ flies (p<0.001; Fig. 4A; Table 1) and for about 2.5 h in UAS-p38b^DNS;Pdf-Gal4/+;+ flies as compared to controls (p<0.001; Fig. 4B; Table 1). Again, no higher fraction of arrhythmic flies was observed (Table 1).

The high number of arrhythmic flies after knockdown of p38b, which was not observed with any of the other constructs, points to putative off-target effects of the p38b^RNAi construct. Off-target effects can never be excluded with the RNAi technology and are more likely to occur if the RNAi construct is expressed in many neurons as was the case with the tim-gal4 driver. When p38b^RNAi was expressed with pdf-gal4, that drives in only 16 neurons in the brain, the number of arrhythmic flies was significantly lower (χ²-test: χ² = 17.02; p<0.001). Thus, it is most likely that the p38b^RNAi construct has off-target effects causing arrhythmicity in addition to its specific effects on clock speed. To test, whether the molecular clock is still running in DD after knockdown of p38b, we immunostained brains of dicer2;UAS-p38b^RNAi/Pdf-Gal4;+ flies with anti-PER and anti-TIM throughout the circadian cycle on day 4 in DD (Fig. S6). We found that the molecular cycling persisted, just the phase of the oscillation was delayed in comparison to controls, which is consistent with the long period of the flies. We conclude that p38 mainly affects the speed of the clock and has little if any effects on the stability and robustness of the molecular clock cycling.

Since p38b^RNAi knockdown and expression of non-functional p38b cause period lengthening of locomotor free-running rhythms, we next wondered what would happen if we overexpress wildtype p38b (UAS-p38b⁺) in different subsets of clock neurons. Surprisingly, although p38b overexpression might have been expected to give the opposite effect of p38b^RNAi (i.e., short locomotor free-running period), UAS-p38b⁺;Pdf-Gal4/+;+ and UAS-p38b⁺;tim(UAS)-Gal4/+;+ exhibited significantly later evening activity in LD and longer locomotor free-running rhythms in DD that were similar to those of p38b^RNAi and p38b^KD flies (p<0.001 and p<0.001 respectively; Fig. S7 and Table 1). This suggests that there is an optimal level of p38b for provoking locomotor activity rhythms with normal period.

Taken together our results indicate that wildtype levels of functional p38b are required for wildtype timing of evening activity and normal/wildtype free-running rhythms under constant conditions. Furthermore, already p38b knockdown or overexpression restricted to the LN_vs (PDF-neurons) is sufficient to cause free-running rhythms with long period. This is well consistent with the dominant role of the s-LN_vs, in which we found p38 expression, in controlling rhythms under constant darkness (reviewed in [61]). Since the oscillation speed was significantly affected by p38b manipulation, we rather assume a function of p38 MAPK in the core of the clock than in its input pathway.

As shown before, the p38a isoform appeared to be co-expressed in the clock neurons (Fig. S2) raising the question whether p38a has also a possible function within the circadian system. To test this, we down-regulated p38a either in all clock cells (using dicer2;tim(UAS)-Gal4;+) or only in the LN_vs (using dicer2;Pdf-Gal4;+), as done before for p38b. The effectiveness of p38a transgenic RNAi was successfully confirmed via qPCR on w;+;UAS-p38a^RNAi/da-Gal4 fly heads (p<0.001; Fig. S4). Down-regulation of p38a had generally weaker effects than down-regulation of p38b: it did not significantly delay evening activity in LD and, in DD, period was not lengthened as dramatically as after manipulation of p38b protein level (Fig. 5). Nevertheless, χ²-periodogram analysis revealed that both experimental lines (dicer2;tim(UAS)-Gal4/+;UAS-p38a^RNAi/+ and dicer2;Pdf-Gal4/+;UAS-p38a^RNAi/+) had significantly longer free-running periods than the respective controls (p<0.001 and p<0.05 respectively; Fig. 5A,B; Table 1). This result strongly argues for a clock-related role for p38a besides p38b.

After we found that down-regulation of p38b or p38a significantly affected the flies' free-running rhythms, we aimed to test whether a complete loss of either isoform does affect rhythmicity in a similar way. To our surprise this was not the case.

Although p38b^Δ45, a p38b null mutant, displayed a slightly enhanced percentage of arrhythmic flies in DD, this was also the case for its precise excision line p38b^pex41, indicating some background effect on rhythmicity (Table 1). Additionally, both lines showed similar activity profiles under LD conditions and did not differ in their free-running period (Fig. 6A; Table 1). Even if p38a null mutants, p38a^Δ1, showed a later onset of evening activity and a higher fraction of arrhythmic flies than the w¹¹¹⁸ controls (Fig. 6B and Table 1), the free-running period of the rhythmic flies was not different from that of the controls. The higher percentage of arrhythmic flies might be associated with the prominent role of p38a in immune stress response [39]–[40], inflammation [26], [62] and lifespan [27]. Therefore flies lacking this isoform might be less healthy and display disrupted behavioral rhythms.

Our results suggest that the two isoforms might replace each other under certain conditions. According to Han et al. [26] p38a and p38b appear to have partial functional redundancy, because both isoforms are similarly activated in response to stress-inducing or inflammatory stimuli in cell culture experiments. This compensatory mechanism may be vital for the flies, when one of the two isoforms lacks completely. But the compensatory mechanism may be elusive when one isoform is only down-regulated in specific neurons that are not necessary for survival (e.g. the clock neurons): Lengthened free-running rhythms in DD just occurred, when either p38b or p38a levels in clock neurons were reduced, but not completely absent from the entire fly. We therefore suppose that either isoform overtakes the clock specific function of the other one only in its complete absence. As p38a mRNA levels were not increased in p38b^Δ45 flies and p38b mRNA levels were not elevated in p38a^Δ1 (Fig. S8), normal wildtype p38a or p38b levels seem to be sufficient to drive circadian rhythms in complete absence of the other isoform.

If our hypothesis is true, p38b^Δ45;p38a^Δ1 double mutants should show long free-running periods. Unfortunately, the combination of both null alleles turned out to be lethal. Similarly, double mutants lacking the p38a gene and carrying a hypomorphic p38b allele (p38b^Δ25;p38a^Δ1) were hardly viable. Furthermore, flies that hatched had a very short life-span dying 3–6 days after emergence of the pupa, making it hard to investigate their free-running rhythms. Nevertheless, we were able to record the locomotor activity of two p38b^Δ25;p38a^Δ1 mutants for 5–6 days, which were entrained to LD 12∶12 during pupal stage and immediately transferred into DD after eclosion (Fig. 6C). These flies free-ran with a long period until they died. In addition, we could simultaneously down-regulate p38a and p38b in TIM-positive neurons (dicer2;UAS-p38b^RNAi/tim(UAS)-Gal4;UAS-p38a^RNAi/+). Such flies exhibited also significant longer free-running periods in DD than the relevant controls (p<0.001; Table 1).Together, our findings strongly indicate that both p38 isoforms are involved in the control of locomotor activity rhythms under constant conditions and that they can partly replace each other.

Delayed evening activity and long free-running rhythms are often associated with a delayed nuclear entry of PER and TIM, an event in the molecular cycle which is mainly regulated via phosphorylation of PER by proline-directed kinases and SGG [12] as well as of TIM by SGG [63]. To see whether the nuclear entry of PER is affected by p38 MAPK, we immunostained respective controls and flies, in which the dominant-negative form of p38b was expressed in the LN_vs (UAS-p38b^DN-S;Pdf-Gal4/+;+), in 1-hour intervals in LD and quantified the amount of nuclear PER in the s-LN_vs and l-LN_vs (Fig. 7). We chose UAS-p38b^DN-S;Pdf-Gal4/+;+ since the delay in evening activity under LD conditions was most prominent compared to other p38 mutant strains. Interestingly, we found a significant delay of nuclear entry of PER in both types of clock neurons that perfectly matched the delayed evening activity.

There are several ways, how p38 could influence the phosphorylation degree of PER and this way influence the efficiency and speed of nuclear translocation: p38 may directly phosphorylate PER or it may activate or inhibit already known key kinases of PER. Accordingly, p38 was shown to phosphorylate and activate CK2 [34]–[35] making it possible that p38 lengthens the period of the molecular oscillations via CK2 finally leading to a delayed nuclear translocation of the PER-TIM heterodimer. Alternatively or in addition, p38 may work on phosphatases that reduce phosphorylation. Previous studies revealed that both, p38 [64] and CK2 [65], stimulate the activity of the protein phosphatase 2A (PP2A) in mammalian fibroblasts. PP2A on the other hand was shown to dephosphorylate and stabilize PER, thereby promoting PER's nuclear translocation in Drosophila clock cells [66]. Consequently, reduction of PP2A activity resulted in long free-running periods, the same phenotype we observed after manipulation of p38 levels.

To test whether p38b affects the degree of PER phosphorylation, we performed Western Blots on head extracts of flies, in which the dominant-negative p38b transgene (UAS-p38b^DN-S) was driven in all clock cells including the photoreceptor cells (in LD 12∶12). This time, we did not use Pdf-gal4, since Western Blots mainly reflect PER oscillations of the compound eyes (the oscillations of the 150 PER-expressing clock neurons can barely be seen behind the oscillations of the ∼1600 PER-expressing photoreceptor cells). Indeed, PER seemed to be less phosphorylated in flies with impaired p38b signaling (Fig. 8A). For a better comparison we repeated the Westerns blotting control and experimental flies for each ZT side by side (Fig. 8B). We found that PER was clearly less phosphorylated in the flies with impaired p38b signaling at all time points. This was most evident during the night being well consistent with the postulated high activity of p38 MAPK during darkness. We conclude that p38 promotes PER phosphorylation during the night. The lack of this phosphorylation may delay nuclear entry of PER during constant darkness and in this way lengthen the free-running period of the clock significantly.

The next question to ask was, whether p38 can phosphorylate PER directly. PER becomes phosphorylated at multiple sites, some of which could be identified as predicted MAPK target sites [14]. Furthermore, Nemo/NLK, an evolutionarily conserved MAPK-related kinase, was shown to function as a priming kinase phosphorylating PER at the recently identified per-short phospho clusters and thereby stimulating phosphorylation of PER by DBT at several nearby sites [11], [13]. In addition, Ko et al. [12] could show that phosphorylation of serine 661 (Ser661) is a key phospho-signal on PER regulating the timing of PER's nuclear accumulation and that this phosphorylation event can be performed by proline-directed kinase(s), as could be shown for ERK in vitro. Mutant flies with blocked S661 phosphorylation site, display a delay in PER's nuclear entry in pacemaker neurons as well as long behavioral rhythms. Moreover, abolishing phosphorylation at Ser661 also diminishes the extent of hyperphosphorylation of PER in vivo, suggesting that the phosphorylated state of Ser661 regulates phosphorylation at other sites on PER. With Ser657 the authors also identified a phosphorylation target site of SGG, which seems to be phosphorylated in a manner dependent on priming at Ser661. Due to the similar phenotypes on molecular as well as behavioral level of period mutants lacking the phosphorylation site at Ser661 and p38 mutants, we aimed to test whether p38 might also phosphorylate PER. Therefore, we created hexa-histidine tagged p38b (His₆-p38b) and two GST tagged, truncated PER isoforms carrying GST amino-terminal fused either to amino acids 1–700 (GST-PER^1–700) or to amino acids 658–1218 (GST-PER^658–1218), and performed in vitro kinase assays. For visualization of protein phosphorylation, samples were subsequently separated on 9% urea-polyacrylamide gels followed by Coomassie staining. We chose urea-PAGE for protein separation, since urea does not mask the charge of the protein and therefore leads to longer runs of phosphorylated proteins due to the negative charges of phosphate residues (Fig. 9A, C). As we wanted to confirm the PER signal on the Coomassie gel, two samples of the gel were additionally blotted to nitrocellulose membrane following gel electrophoresis and detected by immunolabeling (Fig. 9B, D). Both, GST-PER^1–700 and GST-PER^658–1218, displayed obvious band shifts after 60 minutes of incubation with His₆-p38b, while substrate controls without kinase did not shift in the appropriate time (Fig. 9A–D). Even if shifts are not extensive they are clearly visible. The appearance of several shifted bands (Fig. 9C, D) indicates that p38 might phosphorylate PER at several sites and thereby prime it for further phosphorylation. This could explain the overall amount of less phosphorylated PER we found in flies with impaired p38b signaling. Indeed, sequence analysis revealed two putative p38 consensus phosphorylation sites (PXS*P) in PER (Fig. S9): Ser661, that was shown to be phosphorylated by a proline-directed kinase and led to long free-running rhythms when mutated [12], and Ser975. To test whether p38 MAPK, which also belongs to the family of proline-directed kinases, phosphorylates PER at one of these sites, we mutated GST-PER^658–1218 by replacing Ser with Gly either at position 661 (S661G), or at position 975 (S975G) or at both positions (S661G/S975G). Radioactive in vitro kinase assays were performed with bacterially expressed and purified GST-p38b together with wild-type and mutant forms of PER as substrates (Fig. 9E, F). Although p38b phosphorylated all forms of PER, phosphorylation was significantly reduced in the two single mutants and even further reduced in the S661/975G double mutant. Thus, we conclude that p38b can phosphorylate PER at S661 and at S975 - at least in vitro. Future studies have to show whether p38 does phosphorylate PER also in vivo at both sites and whether p38 may compete with other kinases at S661 (e.g. Nemo/NLK, ERK). The complex behavioral phenotypes (period-lengthening after down-regulation and overexpression of p38, as well as no effects of null mutations in p38a and p38b) argue for the putative interaction of several kinases in PER phosphorylation at S661.

In summary, our results demonstrate direct effects of p38 on circadian rhythms in behavior as well as on the molecular clock. Besides affecting the phosphorylation degree and nuclear entry of PER, p38 may influence the clock machinery in several ways due to its many putative targets in Drosophila's clock neurons. As we show here, one of the major p38 targets may be PER itself. Altogether, this places p38 in the center of multiple pathways that can affect circadian rhythms. Regarding its known role in transmitting cellular stress responses, p38 MAPK may even act as a factor that integrates responses of the circadian clock and the acute stress system to external stimuli. However, future studies have to reveal the exciting connection between the two systems in more detail.

Flies were raised on a standard cornmeal/agar medium at 25°C in LD 12∶12. To investigate locomotor activity in p38 mutant flies, we recorded two p38 knockout strains: w¹¹¹⁸;+;p38a^Δ1 and yw;p38b^Δ45;+(kindly provided by R. Cagan and A. Vrailas-Mortimer). The latter carries a 1065bp deletion in the coding region of p38b, while w¹¹¹⁸;+;p38a^Δ1 flies completely lack the p38a locus. In addition the precise excision line yw;p38b^pex41;+ (a gift of A. Vrailas-Mortimer) served as control for yw;p38b^Δ45;+. w¹¹¹⁸ flies served as control for the w¹¹¹⁸;+;p38a^Δ1 mutants. Two double mutant strains, p38b^Δ45;p38a^Δ1 and p38b^Δ25;p38a^Δ1 (both provided by A. Vrailas-Mortimer; the latter exhibits a hypomorphic p38b allele) were used to knockout both p38 isoforms; but these turned out to be either lethal or only weakly viable in our hands. Therefore, we could not perform any statistical analysis of their locomotor activity rhythms. For studying p38 knockdown exclusively within the circadian clock, we used two different RNAi lines, w;+;UAS-p38a^RNAi (Vienna Drosophila RNAi Center; #52277) and w;UAS-p38b^RNAi;+ (Vienna Drosophila RNAi Center; #108099), as well as a combination of both (w;UAS-p38b^RNAi;UAS-p38a^RNAi). To restrict RNAi-mediated gene silencing to specific subsets of clock neurons, RNAi lines were crossed to a w;tim(UAS)Gal4;+ (kindly provided by Michael W. Young) as well as a yw;Pdf-Gal4;+ driver line (kindly provided by Jeffrey C. Hall) and combined with a UAS-dicer2;+;+ line (Vienna Drosophila RNAi Center; #60012) to further strengthen RNAi knockdown. In addition yw;Pdf-Gal4;+ and w;tim(UAS)-Gal4;+ flies were used to specifically overexpress wildtype p38b (UASp38b⁺ kindly provided by T. Adachi-Yamada) as well as two non-functional p38b isoforms: a dominant-negative UAS-p38b transgene, UAS-p38b^DN-S (donated by T. Adachi-Yamada), and an UAS-p38b kinase-dead transgene, UAS-p38b^KD(a gift of A. Vrailas-Mortimer). The dominant-negative p38b allele was generated by replacing the Thr184 of the MAPKK target site with Ala leading to a complete loss of enzymatic activity [28]. The UASp38b^KD transgenic line, however, was made by exchanging a Lys residue at 53 in the catalytic domain with Arg [27]. This single amino acid substitution still allows target binding, but blocks kinase activity (A. Vrailas-Mortimer, personal communication). Here we used flies with two UAS-p38b^KD transgenes, a weaker UAS-p38b^KD3 and a stronger UAS-p38b^KD8 (UAS-p38b^KD3/CyO-GFP;UAS-p38b^KD8/TM3Ser-GFP). For studying the expression pattern of p38 within the brain of Drosophila melanogaster a Canton S (CS) wildtype strain was chosen as wildtype control for immunohistochemistry. In addition a p38b-Gal4 enhancer trap line (kindly provided by A. Vrailas-Mortimer) was used in combination with w;+;UAS-GFP^S65T (Bloomington Stock Center, #1522; donated by Karl Fischbach) to express green fluorescent protein (GFP) in the p38b-neurons revealing p38b expression in detail. To analyze PER cycling on Western Blots, we used a w;cry-Gal4;+ driver line (kindly provided by F. Rouyer) to impair p38b signaling in p38b^DN-S-flies.

For in vitro kinase assays, N-terminal hexa-histidine or GST-tagged p38b fusion proteins (His₆-p38b and GST-p38b) were created by first PCR amplifying the full-length p38b open reading frame (ORF) using the cDNA clone as template and following primers: 5′-CCGATCGAAATGTCGCGCAAAATGGCCAAATTC-3′ and 5′-GGCGGCCGCGATTACTGCTCTTTGGGCAGGAGCTCA-3′. After digestion with PvuI and NotI, the PCR product was inserted into the multiple cloning site of E. coli expression vector pH6HTN His₆HaloTag T7 (Promega) and further subcloned as an EcoRI/NotI fragment into the pGEX 4T3 vector (GE Life Sciences). In order to generate recombinant GST-PER fusion construct, two truncated sequences of per, either encoding amino acids 1–700 (PER^1–700) or amino acids 658–1218 (PER^658–1218), were subcloned into the pGEX 6P vector (GE Life Sciences). All constructs were confirmed by DNA sequencing before use.

GST-PER^658–1218S661G (pGEX6P-per^S661G) and GST-Per^658–1218S975G (pGEX6P-per^S975G) constructs were generated by mutagenesis PCR using pGEX6P-per^658–1218 as template. The primers 5′-CTCGTGGACGGGACCCATGGGCCCACTGGCGCCACTG-3′ and 5′-CAGTGGCGCCAGTGGGCCCATGGGTCCCGTCCACGAG-3′ were used to generate GST-pGEX6P-per^S661G and the primers 5′-CTTGACGCCCACCGGGCCCACGCGCTCTCC-3′ and 5′GGAGAGCGCGTGGGCCCGGTGGGCGTCAAG-3′ were used for pGEX6P-per^S975G generation. To generate the double mutant GST-PER^658–1218S661/975G we performed a second mutagenesis PCR using pGEX6P-per^S661G as template and the pGEX6P-per^S975G mutagenesis primers as described above.

Locomotor activity of individual flies was recorded using the Drosophila Activity Monitoring (DAM) System (Trikinetics) as previously described [67]. Briefly, to investigate locomotor behavior 3–7 day old male flies were monitored in LD 12∶12 for 7 days (with a light intensity of 100 lux in the light phase) followed by additional 14 days in constant darkness (DD). In case of p38b^Δ25;p38a^Δ1, flies were entrained in LD 12∶12 during pupal stage and monitored directly after eclosion in DD conditions. All recordings took place under constant 20°C in a climate–controlled chamber. Raw data of individual light beam crosses were collected in 1-minute bins and displayed as double-plotted actograms using ActogramJ [68], a freely available Java plug-in of ImageJ (freely available at http://rsb.info.nih.gov/ij/↗). We generally excluded data of the first experimental day from analysis to exclude side effects of fly handling. For generating average daily activity profiles for single genotypes, first raw data of day 2–7 in LD were averaged for each single fly. Thereafter, single activity profiles were averaged across all entrained flies of each genotype and smoothed by applying a moving average of 11. For determining the individual free-running period (τ) of rhythmic flies, DD data from day 2–12 were analyzed using χ²-periodogram analysis and average period length of each genotype was calculated. To analyze timing of evening activity, raw LD data were converted into 15 minutes bins and evening activity onset was determined after generation of average days for each single fly. Finally, data were averaged across the genotype and tested for statistically significance.

To investigate p38 expression and oscillations in nuclear PER in adult Drosophila brain, 5–10 days old male flies were entrained to LD 12∶12 for at least 4 days and collected at indicated Zeitgeber Times (ZT; ZT0 indicates lights-on and ZT12 lights-off). To analyze nuclear PER and TIM localization under free-running conditions, flies were first entrained to LD 12∶12 for 4 days followed by 4 days in constant darkness and collected 96 hours after lights-on (ZT1) of the last day LD every 4 hours. Time points of collections were afterwards converted into Circadian Time (CT) according to the onset of activity in free-running flies that were monitored in parallel under the same conditions. Hereby, the activity onset of the flies on day 4 in DD is defined as CT0 and their activity offset as CT12. For light pulse (LP) experiments flies were reared in LD12∶12 for 4 days, subsequently transferred to DD and collected at CT6 and CT18 on day 1 in DD right before as well as after 15 minute light-pulse. Flies were fixed in 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB; pH 7.4) with 0.1% Triton X-100 for 2.5 hours. For fixation of flies expressing GFP, no Triton X-100 was used in the PFA solution and fixation time was increased for additional 30 minutes. The fixation step was carried out on a shaker at room temperature and, if necessary, in absence of any light. After fixation flies were rinsed five times for 10 minutes in PB. After dissection 5% normal goat serum (NGS) in PB with 0.5% Triton X-100 was used for blocking samples overnight at 4°C. Next, samples were subsequently incubated with primary antibodies that were diluted in PB with 0.5% Triton X-100, 5% NGS and 0.02% NaN₃ as follows: chicken anti-GFP 1∶1000 (Abcam), rabbit anti-PER 1∶1000 (kindly provided by R. Stanewsky), rat anti-TIM 1∶1000 (kindly provided by I. Edery), mouse anti-PDF 1∶1000 (Developmental Studies Hybridoma Bank; DSHB), guinea pig anti-VRI 1∶3000 (kindly provided by P. Hardin), goat anti-p38 1∶50 (dN20; Santa Cruz Biotechnology), rabbit anti-p38b 1∶100 (Adachi-Yamada et al., 1999; kindly provided by T. Adachi-Yamada) and rabbit anti-phospho-p38 1∶100 (#4631; Cell Signaling Technology). Goat anti-p38 and rabbit anti-p38b are directed against Drosophila p38 and recognize the active (phosphorylated) and inactive (unphosphorylated) forms of p38a and p38b (http://www.scbt.com/datasheet-15714-p38-dn-20-antibody.html↗; T. Adachi-Yamada personal communication). Rabbit anti-phospho-p38 recognizes human p38 only when dually phosphorylated at Thr180 and/or Tyr182 and does not cross-react with phosphorylated forms of neither p42/44 MAPK nor SAPK/JNK. Due to high-sequence-homology of the p38 phospho sites, the antibody recognizes also Drosophila phospho-p38 [32]. Furthermore, its specifity for phospho-p38 has been shown by several studies [69]–[70]. After 24–48 hours primary antibody incubation samples were rinsed five times for 10 minutes in PB with 0.5% Triton X-100 before secondary antibodies were applied. For double or triple immunolabeling Alexa Fluor 488, Alexa Fluor 555 and Alexa Fluor 647 (all from Molecular Probes) were used as secondary antibodies in a dilution of 1∶200 in PB with 5% NGS and 0.5% Triton X-100. After 3 hours at room temperature secondary antibody solution was removed and samples were rinsed five times for 10 minutes in PB with 0.5% Triton X-100. After a final wash step in PB with 0.1% Triton X-100 brains were embedded in Vectashield mounting medium (Vector Laboratories).

The fluorescence signal of immunolabeled brains was visualized using a laser scanning confocal microscope (Zeiss LSM 510 Meta; Carl Zeiss MicroImaging Germany) with a 20× objective. To excite the fluorophores of the secondary antibodies, we used three different diode laser lines 488 nm, 532 nm and 635 nm. In order to avoid bleed through, individual channels were scanned separately, one after another. After confocal stacks of 2 µm thickness were obtained, stacks were subsequently imported into ImageJ to measure staining intensities, to crop the images and to generate overlays. Except of adjustment of brightness and contrast, we performed no other manipulations on the images. To quantify p-p38 staining intensity, both hemispheres of 10 brains per genotype were examined and staining intensity of DN_1as was measured using ImageJ as described previously [71]. In order to investigate nuclear translocation of PER in LN_vs of UAS-p38b^DN-S;Pdf-Gal4/+;+ flies and respective controls, we examined 7 brains per ZT and genotype. This time the parameters area, integrated density and mean grey value of defined regions (nucleus, cell body and respective background) were measured and the corrected total cell fluorescence (CTCF) of nucleus as well as cell body was calculated using following formula: CTCF_Nucleus/Cell = Integrated density_Nucleus/Cell – (Area_Nucleus/Cell×Mean fluorescence_Background). Finally, nuclear signal (CTCF_Nucleus) was normalized to total cell fluorescence (CTCF_Cell) to determine nuclear translocation of PER in s-LN_vs and l-LN_vs.

To analyze p38 mRNA expression, 5–10 days old male adult flies were synchronized by LD 12∶12 for 4 days. On the fifth day flies were collected according to ZTs and quickly decapitated on ice. Total RNA from 5 fly heads per genotype and ZT was extracted using the Quick RNA Micro Prep Kit (Zymo Research). cDNA derived from this RNA (using QuantiTect Reverse Transcription Kit from Qiagen) was used as a template for quantitative real-time PCR (qPCR) in combination with the SensiFAST SYBR No-Rox Mix (Bioline) and one of the following primers: 5′-GCCCGTAGACAAATGGAAGGA-3′ and 5′-AACCTGAGCATACGATGGTGG-3′ for p38a, 5′-GAGATGGTCTTCAGCGAGGT-3′ and 5′-AGCATCATTGAACGGAGAGGG-3′ for p38b and 5′-TCTGCGATTCGATGGTGCCCTTAAC-3′ and 5′-GCATCGCACTTGACCATCTGGTTGGC-3′ for α-tubulin.

5–10 days old flies were entrained to LD 12∶12 for at least 4 days and collected every 2 hours. To analyze PER cycling, 25 heads of male flies per ZT were homogenized in protein extraction buffer (20 mM HEPES pH 7.5; 100 mM KCl; 5% glycerol; 10 mM EDTA; 0.1% Triton X-100; 20 mM β-glycerophosphate; 0.1 mM Na₃VO₄ pH 11) containing a protease inhibitor cocktail (cOmplete Mini EDTA-free; Roche) and loaded onto a 6% gel. SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose paper were performed according to standard immunoblotting protocols. To minimize differences due to variations in gel electrophoresis and protein blotting, samples of flies with altered p38 levels and respective controls were run and blotted to membrane simultaneously and repeated 4 times. For visualizing daily PER cycling, membranes were incubated in primary and secondary fluorescent antibodies which were diluted in tris-buffered saline with 0.1% Tween-20 (TBST) as follows: rabbit anti-PER 1∶10000 (kindly provided by R. Stanewsky), Alexa Fluor goat-anti-rabbit 680 1∶5000 (Invitrogen). Fluorescent signals were detected using the Odyssey Imaging System (Li-cor Bioscience).

To express His₆-p38b, the expression construct was introduced into BL21(DE3)pLYSs competent E. coli cells (Promega) and protein expression was induced at an optical density of ∼0.5 (OD₆₀₀) with 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3 hours at 37°C. After induction cells were pelleted, washed in phosphate-buffered saline (PBS) and pellet was frozen once overnight. Thawed lysate was then solubilized in lysis buffer (50 mM NaH₂PO₄; 300 mM NaCl; 10 mM imidazole; 1 mM PMSF; 10 µg/ml leupeptin; pH 8.0) containing protease inhibitor cocktail (complete Mini EDTA-free; Roche) and sonicated 5×5 seconds with short pauses on ice. After sonication Triton X-100 was added to a final concentration of 1% and lysate was centrifuged at 10000 g for 30 minutes at 4°C. Purification of His₆-p38b protein kinase from supernatant was subsequently carried out by column chromatography using His-Select Nickel Affinity Gel (Sigma) according to manufacturer's protocol. Finally, His₆-p38b was concentrated and elution buffer (50 mM NaH₂PO₄; 300 mM NaCl; 250 mM imidazole) was exchanged by several centrifugation steps (3 minutes at 4000 rpm) using Amicon Ultra centrifugal filter units (MWCO 30 kDa; Sigma) and PBS. For expression of the various GST-PER fusion proteins and GST-p38b, E. coli DH5α containing the appropriate expression plasmids were grown at 37°C to an optical density of 1.2 (OD₆₀₀). Expression was induced by adding IPTG to a final concentration of 0.1 mM accompanied by a reduction of the incubation temperature to 25°C. After 4 hours bacteria were harvested by centrifugation and solubilized in lysis buffer (137 mM NaCl; 2.7 mM KCl; 10 mM Na₂HPO₄; 1.8 mM KH₂PO₄; 100 mM EDTA; 1% Triton X-100; pH 7.5) supplemented with protease inhibitors (Roche Complete Cocktail and 1 mM PMSF). Resuspended cells were then lysed by sonication and the lysate was cleared by 30 minutes centrifugation at 15000 g and 4°C. After centrifugation, lysates were incubated at 4°C overnight on a rotary wheel with 1.5 ml glutathione sepharose 4B beads (GE Life Sciences) to bind the fusion proteins. The beads were then transferred to a 10 ml Polyprep column (Biorad) and washed once with lysis buffer and thrice with wash buffer (50 mM Tris; 100 mM EDTA; 0.1% Tween-20; pH 7.5). The fusion protein was then eluted from the GSH-Sepharose beads using a 100 mM glutathione solution adjusted to pH 7.5 with 1 M TrisHCl (pH 8.8). Finally the eluate was dialyzed in 5 mM TrisHCl (pH 7.5) for 48 hours and stored at −80°C. To perform non-radioactive phosphorylation assays, 5 µM substrate (GST-PER^1–700 and GST-PER^658–1218) was incubated in kinase buffer (50 mM Tris-HCl; 5 mM DTT; 30 mM Mg²⁺; 0.1 mM Na₃VO₄) containing 1 mM ATP at 30°C. Kinase assays were initiated by the addition of 1 µM His₆-p38b and stopped directly after, 30 minutes or 60 minutes after kinase addition by adding equal amount of 2× urea-loading buffer (9 M urea; 20 mM Tris; 190 mM glycine; 1.5 mM EDTA pH 7.5; 1 mM DTT; 0.016% brom phenol blue). After one additional hour incubation at room temperature, protein separation was examined using urea-PAGE and visualized by Coomassie staining, except of some samples that were cut before Coomassie treatment and immunoblotted as described in the western blot section.

Radioactive in vitro kinase reactions were conducted in standard kinase buffer (20 mM HEPES pH 7.6, 20 mM MgCl₂, 10 mM β-glycerophosphate, 0.5 mM Na₃VO₄) containing 5 µCi γ-³²P-ATP per reaction. 20 µg of GST-p38b kinase and of the indicated GST-PER fusion protein or GST as a control were added to each reaction and incubated at 30°C for 30 minutes. The proteins were then separated by SDS-PAGE and phosphorylation was detected by autoradiography. Gels were stained with Coomassie brilliant blue to visualize total protein amounts in the various samples. Relative levels of phosphorylation were quantified using the open source software Fiji [72].

All data were tested for normal distribution by a one-sample Kolmogorov–Smirnov test (Lilliefors). Normally distributed data were statistically compared by a one-way analysis of variance (ANOVA) followed by a post-hoc test with Bonferroni correction for pairwise comparison. Not normally distributed data were compared by a Kruskal–Wallis test followed by Wilcoxon analysis (Systat 11, SPSS, Chicago, IL; USA) with Bonferroni correction. Data were regarded as significantly different at p<0.05 and as highly significant at p<0.001.